The instant invention is related to the field of thermophilic microorganisms the stable gene transfection in and protein expression thereof, and in the genetic thermostabilization of proteins.
Extreme thermophilic microorganisms such as Thermus, thrive in high-temperature environments that are lethal to other known forms of life. Fortunately, apart from their higher growth temperature requirement, they can be handled in the laboratory much like E. coli. Enzymes from thermophiles are thermostable and are therefore used in industrial processes that benefit from a high reaction temperature. Also, these enzymes have become widely used in molecular genetic research, for example, in the development and application of the polymerase chain reaction (PCR).
One area of particular interest in the field of thermophile research is the determination of molecular mechanisms underlying enzymatic thermostability. Ultimately, a better understanding of this phenomenon will allow mesophilic proteins to be rationally converted to thermostable proteins for industrial applications. Many groups have attempted to engineer thermostability into proteins through in vitro rational design approaches (Perry and Wetzel, 1984, Science 226:555-557; Sauer, at al., 1986, Biochemistry 25:5992-8; Pantoliano, et al., 1987, Biochemistry 26:2077-82; Meng, et a/., 1993, Bio/Technology 11:1157-1161) or through homology comparison and domain fusion of related proteins (Onodera, et al., 1991, J Biochem 109:1-2; Barany, et al., 1992, Gene 112:3-12; Politz, et al., 1993, Eur J Biochem 216:829-34; Lee, et al., 1993, J Bacteriol 175:5890-8). These approaches, however, often require either a three dimensional protein structure or a series of related proteins. For proteins which have not been well characterized, random mutagenesis can be a powerful tool if the proper selection or screen can be applied (Matsumura and Aiba, 1985, J Biol Chem 260:15298-15303; Liao, et al., 1986, Proc Natl Acad Sci USA 83:576-580; Kajiyama and Nakano, 1993, Biochemistry 32:13795-9; Arnold, 1993, Faseb J 7:744-9). The instant invention provides a genetic process for the insertion of exogenous protein coding sequences into, and direct selection of thermostable variants of mesophilic enzymes. Other xe2x80x9cthermo-geneticxe2x80x9d processes, were attempted by Liao (Liao et al., 1986, Proc. Natl. Acad. Sci. USA 83: 576-580), EP Patent application 0 138 075, and by Matsumura (Matsumura et al., 1985, J. Biol. Chem. 260: 15298-15303).
The concept of xe2x80x9cthermo-geneticsxe2x80x9d consists of a method for introducing a gene of interest into a thermophile followed by a temperature-shift to select for temperature-resistant mutations in the corresponding protein of interest. The model thermo-genetic systems (EP Patent application 0 138 075; Matsumura, 1985; Liao, 1986) used the mesophilic kanamycin-resistance gene (kan) on a multicopy plasmid in the moderate thermophile Bacillus stearothermophilus. The kan gene was first introduced into B. stearothermophilus at the lowest permissible temperature of growth, 47xc2x0 C. Two consecutive thermal shifts, first to 63xc2x0 C. and then to 69xc2x0 C., resulted in two corresponding thermo-stabilizing mutations, producing the double mutant allele, designated here as kantr2. At this point the upper limit for permissible growth had been reached, creating a barrier to further selections for temperature-resistant mutations. Matsumura also performed a related series of experiments generating the same two mutants in parallel (Matsumura et al., 1986, Nature 323: 356-358) and later showed they could be combined with an additive result.
While other host-vector systems have been developed for Thermus thermophilus, a closely related thermophile, they all have deficiencies in their ability to be used in a thermostabilization process and in stable integration of exogenous genes into Thermus. Two plasmid-based systems use multicopy plasmids with an unstable copy number which can interfere with mutant selection (Mather and Fee, 1992, Appl Environ Microbiol 58:421-425; Lasa, et al., 1992, J Bacteriol 174:6424-6431). The multicopy nature of these systems do not ideally lend themselves to thermostabilization of genes since many copies of the gene of interest are present, and can mask any desired mutations which may occur. In addition, reports with plasmid-based systems in Thermus indicate that the plasmids are very unstable, that copy number varies widely, and that gene duplication and amplification can occur, making them very difficult to use. Another approach which used an insertional mutagenesis system was developed by Lasa et. al. (Lasa, et al., 1992, Molec Microbiol 6:1555-1564) but unfortunately caused a debilitating phenotype in the host organism.
In Lasa""s insertional mutagenesis system, the kantr2 was inserted in single copy into a highly-expressed (slpA) region of the chromosome for use in chromosomal insertion strategy (Lasa et al. 1992a, J. Molec. Microbiol. 6, 1555-1564; Lasa et al. 1992b, J. Bacteriol. 174, 6424-6431). This system used the slpA gene which codes for an abundant cell surface protein and therefore was likely to be highly expressed. A high expression site was originally a logical choice for testing the feasibility of a single-copy system. Unfortunately, insertion into slpA results in debilitating growth and morphology phenotypes making it difficult to use the plasmid system.
References which define the background of the invention, but which are not necessarily prior art to the instant invention are as follows. The references cited herein above and below are hereby incorporated by reference in their entirety.
Sen and Oriel (1990) Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus, FEMS Microbiology Letters 67:131-134, teach the use of the Streptococcus transposon Tn916, carrying tetracycline resistance for conjugal transfer into Thermus aquaticus via Bacillus subtilis. This was found to be effective at 48xc2x0 C. and 55xc2x0 C. The actual insertion site is unknown.
Koyama et al. (1990) A plasmid vector for an extreme thermophile, Thermus thermophilus, FEMS Microbiology Letters 72:97-102, teach a Thermus-E. coli shuttle vector carrying a tryptophan synthetase gene (trpB). This cryptic plasmid pTT8, was able to transform Thermus thermophilus. The authors point out that a plasmid vector carrying trpBA was not suitable for selection since the cloned DNA fragment recombined with the chromosomal counterpart at high frequency.
Koyama and Furukawa (1990) Cloning and Sequence Analysis of Tryptophan Synthetase Genes of an Extreme Thermophile, Thermus thermophilus HB27: Plasmid Transfer from Replica-Plated Escherichia coli Recombinant Colonies to Competent T. thermophilus Cells, J. of Bacteriology 72:3490-3495, disclose nucleotide sequences for trpBA genes, their use in plasmids and expression in E. coli under the control of the lac promoter.
Koyama et al. (1986) Genetic Transformation of the Extreme Thermophile Thermus thermophilus and of Other Thermus spp., J. of Bacteriology 166:338-340, discuss the conditions for optimal transformation with exogenous DNA. The use of Thermus thermophilus HB27 did not require chemical treatment to induce competence, although the addition of Ca+2 and Mg+2 was optimal. The optimal conditions were found to be 70xc2x0 C. with a 60 minute incubation, pH 6 to 9.
Borges and Bergquist (1993) Genomic Restriction Map of the Extremely Thermophilic Bacterium Thermus thermophilus HB8, J. of Bacteriology 175:103-110, teach the use of Thermus thermophilus HB8, which carries two cryptic plasmids, pTT8 and pVV8 was examined. A genomic restriction map was generated, 16 genes located on the map.
Matsumura et al. (1984) Enzymatic and Nucleotide Sequence Studies of a Kanamycin-Inactivating Enzyme Encoded by a Plasmid from Thermophilic bacilli in Comparison with That encoded by Plasmid pUB110, J. of Bacteriology 160:413-420, teach the a Kanamycin resistance gene from a thermophilic bacteria plasmid pTB913 was found to differ by only one base pair in the middle of the gene, from that of a mesophilic Staphylococcus aureus plasmid pUB110. The change was a cytosine(pUB) to adenine(pTB) at base position +389, which led to a threonine to lysine change at position 130.
Matsumura and Aiba (1985) Screening for Thermostable Mutant of Kanamycin Nucleotidyltransferase by the Use of a Transformation System for a Thermophile, Bacillus stearothermophilus, J. of Biological Chemistry 260:15289-15303, disclose a structural gene for kanamycin nucleotidyltransferase that was cloned into the single-stranded bacteriophage M13 and then subjected to hydroxylamine mutagenesis. The mutagenized gene was then recloned into a vector plasmid pTB922 and used to transform Bacillus stearothermophilus to select for improved enzyme thermostability. A temperature shift from 55xc2x0 C. to 61xc2x0 C. was used for selection. Two types of mutations were found, at position 80 an aspartate to tryptophan, and at position 130 a threonine to lysine. These were found stable up to 65xc2x0 C. The kan gene came from pUB110.
Matsumura et al. (1986) Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein, Nature 323:356-358, teach improved thermostability of kanamycin nucleotidyltransferase (KNTase) was shown to be due to the Asp80 to Tyr(Y80) and Thr130 to Lys(K130) mutation. This also correlated with increased resistance to proteolysis. Catalytic activity was also measured at various temperatures and it was found that the activities deteriorate slightly as thermostability increases, but the optimal temperature is shifted upwards. It is thought that increased hydrogen bonding and hydrophobic interactions act as forces to stabilize the enzyme.
Liao et al. (1986) Isolation of a thermostable enzyme variant by cloning and selection in a thermophile, PNAS USA 83:576-580, teach a kan gene transferred via shuttle vector into B. stearothermophilus and selected for at 63xc2x0 C. The shuttle plasmid was passed through the E. coli mutD5 mutator strain and introduced by transformation. The vector combined the kan gene from pUB110 with a putative thermostable origin of replication from pBST1, isolated from a kanamycin-sensitive strain NRRL1102.
Lasa et al. (1992a) Development of Thermus-Escherichia Shuttle Vectors and Their Use for Expression of the Clostridium thermocellum celA Gene in Thermus thermophilus, J. of Bacteriology 174:6424-6431, teach the self-selection of undescribed origins of replication from cryptic plasmids from uncharacterized Thermus spp. and Thermus aquaticus are isolated and cloned into E. coli vectors. Plasmids were constructed with these origins, pLU1 to pLU4 from T. aquaticus, and pMY1 to pMY3 from Thermus spp. The plasmids then had a modified form of the cellulase gene (celA) from Clostridium thermocellum and were expressed in E. coli with the signal peptide from the S-layer gene from T. thermophilus. Transformation back into T. thermophilus allowed for expression at 70xc2x0 C.
Lasa et al. (1992b) Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8, Molecular Microbiology 6:1555-1564, teach the transcription and translation signals from the slpA gene from Thermus thermophilus HB8 used to express a thermostable kan gene. After 48 hours at 70xc2x0 C., two isolates were obtained.
Faraldo et al. (1992) Sequence of the S-Layer Gene of Thermus thermophilus HB8 and Functionality of Its Promoter in Escherichia coli, J. of Bacteriology 174:7458-7462, disclose the S-layer gene slpA, sequenced and the function in E. coli described.
Mather and Fee (1992) Development of Plasmid Cloning Vectors for Thermus thermophilus HB8: Expression of a Heterologous, Plasmid-Borne Kanamycin Nucleotidyltransferase Gene, Applied and Envior. Microbiology 58:421-425
A plasmid cloning vector is disclosed which uses the kan gene inserted randomly into a cryptic multicopy plasmid (pTT8) isolated from T. thermophilus. 
Nagahari et al (1980) Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli, Gene 10:137-145, describe the Thermus thermophilus leu locus cloned into E. coli and expressed. The plasmid pBR322-T.leu hybrid plasmid was constructed to encode the xcex2-IPM dehydrogenase activity (leuB), the optimal temperature of which was 80xc2x0 C. Experiments suggest that there is a promoter that may be used in E. coli. 
Tanaka et al. (1981) Cloning of 3-Isopropylmalate Dehydrogenase Gene of an Extreme Thermophile and Partial Purification of the Gene Product, Biochem. 89:677-682, demonstrate the cloning into E. coli , protein production, and heat-treatment purification of Thermus thermophilus 3-IPM.
Croft et al. (1987) Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli, Molec. Gen. Genet. 210:490-497, describe the promoter for the leu BCD genes in Thermus thermophilus HB8. The structural similarity with known leu genes is examined. Thermus DNA failed to complement E. coli leuA mutants. Perhaps leuA is not functional.
Yamada et al. (1990) Purification, Catalytic Properties, and Thermal Stability of Threo-Ds-3-Isopropylmalate Dehydrogenase Coded by leuB Gene form an Extreme Thermophile, Thermus thermophilus Strain HB8, J. Biochem. 108:449-456, demonstrate the product of leuB from Thermus thermophilus as expressed in E. coli by plasmid. The enzyme was purified using heal treatment.
Kirino and Oshima (1991) Molecular Cloning and Nucleotide Sequence of 3-Isopropylmalate Dehydrogenase Gene (leuB) from an Extreme Thermophile, Thermus aquaticus YT-1, J. Biochem. 109:852-857, here the gene encoding T. aquaticus leuB was cloned into E. coli and expressed.
Imada et al. (1991) Three-dimensional Structure of a Highly Thermostable Enzyme, 3-Isopropylmalate Dehydrogenase of Thermus thermophilus at 2.2 xc3x85 Resolution. J. Mol. Bio. 222:725-738, describe the 3D structure of IPMDH from Thermus thermophilus has been determined and refined to 2.2 xc3x85 resolution. The dimeric form of IPMDH is crucial to function.
Onodera et al. (1991) Crystallization and Preliminary X-Ray Studies of a Bacillus subtilis and Thermus thermophilus HB8 Chimeric 3-Isopropylmalate Dehydrogenase, J. Biochem. 109:1-2, teach a chimeric gene fusing the Bacillus subtilis and Thermus thermophilus genes encoding for IPMDH, cloned into E. coli. 
Liao et al., European Patent Application 0 138 075 A1 published 24.04.85, discloses the use of plasmids for transforming thermophilic bacteria. A method for isolating thermostable promoters, a method for selecting thermostable variants of gene products of cloned genes in thermophilic hosts using the plasmids of the invention. Such plasmids as:
pBST1, 80 kb cryptic single copy plasmid isolated from B. stearothermophilus at 70xc2x0 C.
pBST2, 1.4 kb, OR of pBST1 and kanR of pUB110, grows at 70xc2x0 C., kan up to 47xc2x0 C. 3 copy.
pBST2-6, pBST2 with oligonucleotide linker to form HindIII site. kan less than 55xc2x0 C.
pBST2-6TK, variant of pBST2-6, kan activity up to 66xc2x0 C.
pSHW9, chloramphenicolR CAT from pC194, in pBR322, ampR, pBR322 OR+pC194.
pBST8, pBST2-6+pSHW9, xe2x80x9cShuttle vectorxe2x80x9d
pCV1, pSHW9 with promoter substituted by polylinker site.
pCV3, pCV1 missing NarI site.
pBST110, pCV3+pBST2-6, shuttle vector, no CAT production.
pRMS10, shotgun clone into pBST110, a promoter from B. stearothermophilus. 
Lacey, R. W. and I. Chopra. Genetic studies of a multiresistant strain of Staphylococcus aureus. J. Med. Microbiol. 7: 285-297, 1974, describes plasmid PUB110 which contains the Kan gene.